PROJECT SUMMARY/ABSTRACT Prescription of opioids for treatment of pain has nearly quadrupled from 1999 to 2014 leading to an epidemic in addiction and overdose deaths in the United States. Morphine and its synthetic and more potent counterpart, fentanyl, bind to opioid receptors in the peripheral and central nervous system, producing feelings of sedation and euphoria in addition to analgesia. Opioid-induced respiratory depression (OIRD) caused by the activation of opioid receptors on neurons in the brainstem respiratory centers may lead to cardiorespiratory collapse and ultimately death. Naloxone, a competitive opioid receptor antagonist used to treat opioid overdose, blocks the effects of opioids but may cause rapid and severe opioid withdrawal, which is itself a medical emergency. There is substantial inter-individual variation in response to opioids, resulting in variation in sensitivities to opioid overdose and treatment responses. However, the collection of factors and underlying mechanisms that determine the variable responses to opioids remain unknown. The long-term goal is to define the biological basis of opioid overdose risk and to discover safe and effective novel reversal agents. The overall objective of this proposal is to define the molecular mechanisms underlying individual variability in respiratory responses to opioids that indicate specific sensitivities to opioids and pharmacological alternatives to naloxone. Preliminary studies using the founders of the advanced, high-diversity mouse populations, the Collaborative Cross (CC) and Diversity Outbred (DO), have revealed strain differences in opiate lethality and respiratory sensitivity modeling the individual variability to OIRD in humans. The overall objective will be attained by pursuing three specific aims: 1) Map genetic loci that underlie the variability in morphine and fentanyl sensitivity in the DO mouse population; 2) Define the in depth physiological components of OIRD in CC mice using PiezoSleep and plethysmography; and 3) Define the mechanisms that underlie the variable response to morphine and fentanyl by profiling the brainstem transcriptome in CC mice. Under the first aim, quantitative trait loci for survival time, recovery time and depth or respiratory depression due to morphine or fentanyl treatment will be mapped in the DO population. Under the second aim, comprehensive respiratory phenotyping will be performed in nave, morphine-treated and fentanyl-treated CC mice. Under the third aim, expression QTL in the CC mice will be mapped, gene co-expression network modeling will be performed, and gene expression will be correlated with phenotypic variation. The proposed research is innovative because it represents a new and substantive departure from the status quo by using methods that enable unbiased discovery of new pathways and mechanisms of opioid overdose vulnerability and remediation. The proposed study is significant because understanding the mechanisms that underlie the variability in the respiratory response to opioids will address the opioid crisis both through prediction of individuals in whom opioids are contraindicated due to overdose vulnerability and through identification of targets for opioid receptor-independent reversal agent development.